Human Papilloma Viruses (HPVs) are implicated in the etiology of cervical cancer, the fifth most common cancer worldwide and the second cause of cancer-related death in women. If also other HPV-related cancers are taken into account, up to 10% of the worldwide mortality due to cancer is linked to HPVs. HPVs are double stranded circular DNA viruses of about 8 kilobases. Until now more than 60 genotypes have been described of which several are associated with cancer.
HPV-DNA can be found in cervical dysplastic lesions and in cervical carcinomas in which the percentage of HPV positivity increases up to 99% when the lesions progress towards malignancy. The most important HPV types associated with cervical carcinoma are HPV16 and 18 of which HPV16 alone accounts for more than 50% of the HPV positive cervical carcinomas.
The DNAs of several HPVs have been sequenced. The DNA open reading frames can be divided into early regions (E) and late regions (L). The E regions are coding for proteins needed for virus replication and transformation. The L regions encode viral capsid proteins. The E6 and E7 proteins are involved in the pathogenesis of HPV-induced abnormal cell proliferation and these genes are expressed in tissue or tumor cells obtained from cervical cancers associated with HPV infection.
In addition, the E6 and E7 genes of HPV16 and HPV18 are capable of inducing epithelial cell transformation in the cell culture without the presence of other HPV genes indicating that at least part of the stimulation of cell proliferation caused by HPV infection is due to the E6 and E7 viral proteins.
Cytotoxic T lymphocytes (CTL) are of crucial importance in the resistance against virus infections and the immune surveillance against virus-induced tumors (reviewed by Kast and Melief, 1991). CTL specific for viruses or virus-induced tumors recognize short viral protein-derived peptides, of about 9 amino acids in length, that are bound to the antigen presenting groove of major histocompatibility complex (MHC) class I molecules (reviewed by Kast and Melief, 1991). Recently, in several virus systems vaccination with peptides recognized by antigen-specific CTL was shown to prevent lethal virus infections and to delay tumor growth in mice (reviewed by Kast and Melief, 1991, and by Reinholdsson-Ljunggren et al., 1992).
We have succeeded in the identification of viral peptides that bind to the groove of MHC class I molecules by using the antigen processing defective cell line 174CEM.T2 generated and provided by P. Cresswell (see Salter and Cresswell, 1986). This cell line expresses the human MHC class I HLA-A2.1 and HLA-B5 alleles of which only the HLA-A2.1 molecules are expressed as partly empty and unstable molecules that can be stabilized on the cell surface with exogenously added peptides. If incubation with peptide results in an increase in the cell surface expression of this MHC molecule, this implies that the peptide binds to the groove of the HLA-A2.1 molecule and is therefore a possible candidate to be recognized by CTL. The HLA-A2.1 molecule is the most frequent HLA molecule present in the Western European Caucasoid population. About 50% of this population expresses this allele.
Using the amino acid sequence of the E6 and E7 proteins of HPV16 and HPV18 (Seedorf et al., 1985) we generated all possible nonapeptides (i.e. 9 amino acid long peptides) overspanning the entire E6 and E7 region. Every amino acid was used as a start amino acid for these 9-mer peptides. Every peptide was subjected individually to the above test to determine its capacity to bind to the HLA-A2.1 molecule. With respect to HPV16, we identified in total 10 peptides in the HPV16 E6 region and 8 in the HPV16 E7 region which bound to the HLA-A2.1 molecule in the above test. With respect to HPV18, in total 9 peptides in the HPV18 E6 region and 5 in the HPV18 E7 region were identified in the above test to bind to the HLA-A2.1 molecule. This implies that important candidate peptides of HPV16 and HPV18 for use as a vaccine in HLA-A2.1 positive humans have been identified.
By using a second approach, we succeeded to expand the list of HLA-A2.1 binding HPV peptides a little further and to determine HPV16 E6 and E7 peptides binding to other HLA molecules, viz. to the HLA-A1, HLA-A3.2, HLA-A11.2 and HLA-A24 molecules. Said second approach consisted of a competitive immunochemical peptide-MHC binding assay using purified class I molecules and radiolabeled consensus peptides.